Cartilage Proteoglycan Release Assay

Billington, Caron

doi:10.1385/1-59259-046-2:451

Cited by 8 publications

(9 citation statements)

References 3 publications

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“…Likewise, few published protocols explicitly address media samples. Mort and Roughley (2007) instruct that standards for media sGAG analysis should be prepared in the culture medium, but other protocols (Billington, 2001; Hughes et al , 2003; Shingleton, 2003) instruct that phosphate buffer or water should be used for standards and, if necessary, to dilute samples. Our results indicate the that the latter practice could lead to inaccurate results.…”

Section: Discussionmentioning

confidence: 99%

Fact versus artifact: Avoiding erroneous estimates of sulfated glycosaminoglycan content using the dimethylmethylene blue colorimetric assay for tissue-engineered constructs

Zheng

Levenston²

2015

eCM

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Section: Discussionmentioning

confidence: 99%

Fact versus artifact: Avoiding erroneous estimates of sulfated glycosaminoglycan content using the dimethylmethylene blue colorimetric assay for tissue-engineered constructs

Zheng

Levenston²

2015

eCM

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“…DNA content was assayed with a QuantiTPicoGreen kit (Thermo Fisher, Waltham, MA) and measured on a microplate reader (Molecular Devices, Sunnyvale, CA) with excitation at 488 nm and absorption at 525 nm. GAG content was analyzed using a dimethylmethylene blue (DMMB) assay with modifications for alginate and media measurements, and normalized by DNA content. Statistics on normalized total GAG content were calculated using a one‐way ANOVA test and multiple t tests as described in the Gene Expression Analysis section.…”

Section: Methodsmentioning

confidence: 99%

Effects of cell type and configuration on anabolic and catabolic activity in 3D co‐culture of mesenchymal stem cells and nucleus pulposus cells

Ouyang

Cerchiari

Tang

et al. 2016

Journal Orthopaedic Research

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Tissue engineering constructs to treat intervertebral disc degeneration must adapt to the hypoxic and inflammatory degenerative disc microenvironment. The objective of this study was to determine the effects of two key design factors, cell type and cell configuration, on the regenerative potential of nucleus pulposus cell (NPC) and mesenchymal stem cell (MSC) constructs. Anabolic and catabolic activity was quantified in constructs of varying cell type (NPCs, MSCs, and a 50:50 co-culture) and varying configuration (individual cells and micropellets). Anabolic and catabolic outcomes were both dependent on cell type. Gene expression of Agg and Col2A1, glycosaminoglycan (GAG) content, and aggrecan immunohistochemistry (IHC), were significantly higher in NPC-only and co-culture groups than in MSC-only groups, with NPC-only groups exhibiting the highest anabolic gene expression levels. However, NPC-only constructs also responded to inflammation and hypoxia with significant upregulation of catabolic genes (MMP-1, MMP-9, MMP-13, and ADAMTS-5). MSC-only groups were unaffected by degenerative media conditions, and co-culture with MSCs modulated catabolic induction of the NPCs. Culturing cells in a micropellet configuration dramatically reduced catabolic induction in co-culture and NPC-only groups. Co-culture micropellets, which take advantage of both cell type and configuration effects, had the most immunomodulatory response, with a significant decrease in MMP-13 and ADAMTS-5 expression in hypoxic and inflammatory media conditions. Co-culture micropellets were also found to self-organize into bilaminar formations with an MSC core and NPC outer layer. Further understanding of these cell type and configuration effects can improve tissue engineering designs.

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“…Bovine nasal cartilage was cultured as previously described [36]. Briefly, discs (approximately 2 mm in diameter by 1 to 2 mm thick) were punched from bovine nasal septum cartilage; three discs per well in a 24-well plate were incubated overnight in control, serum-free medium (DMEM containing 25 mM HEPES, 2 mM glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin, 2.5 μg/ml gentamicin, and 40 u/ml nystatin).…”

Section: Methodsmentioning

confidence: 99%

“…The viability of cartilage explants was assessed by measurement of lactate dehydrogenase (LDH) in the conditioned medium (CytoTox 96 assay, Promega, Southampton, UK). Hydroxyproline release was assayed as a measure of collagen degradation [37], and glycosaminoglycan release was assayed as a measure of proteoglycan degradation [36]. Collagenase activity was determined by the 3 H-acetylated collagen diffuse fibril assay using a 96-well plate modification [38] and a standard curve and appropriate sample dilutions; one unit of collagenase activity degraded 1 μg of collagen per minute at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Untitled

et al. 2005

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Cartilage destruction in the arthritides is thought to be mediated by two main enzyme families: the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, and enzymes from the ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family mediate cartilage aggrecan loss. Many genes subject to transcriptional control are regulated, at least in part, by modifications to chromatin, including acetylation of histones. The aim of this study was to examine the impact of histone deacetylase (HDAC) inhibitors on the expression of metalloproteinase genes in chondrocytes and to explore the potential of these inhibitors as chondroprotective agents. The effects of HDAC inhibitors on cartilage degradation were assessed using a bovine nasal cartilage explant assay. The expression and activity of metalloproteinases was measured using real-time RT-PCR, western blot, gelatin zymography, and collagenase activity assays using both SW1353 chondrosarcoma cells and primary human chondrocytes. The HDAC inhibitors trichostatin A and sodium butyrate potently inhibit cartilage degradation in an explant assay. These compounds decrease the level of collagenolytic enzymes in explant-conditioned culture medium and also the activation of these enzymes. In cell culture, these effects are explained by the ability of HDAC inhibitors to block the induction of key MMPs (e.g. MMP-1 and MMP-13) by proinflammatory cytokines at both the mRNA and protein levels. The induction of aggrecandegrading enzymes (e.g. ADAMTS4, ADAMTS5, and ADAMTS9) is also inhibited at the mRNA level. HDAC inhibitors may therefore be novel chondroprotective therapeutic agents in arthritis by virtue of their ability to inhibit the expression of destructive metalloproteinases by chondrocytes.

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Cartilage Proteoglycan Release Assay

Cited by 8 publications

References 3 publications

Fact versus artifact: Avoiding erroneous estimates of sulfated glycosaminoglycan content using the dimethylmethylene blue colorimetric assay for tissue-engineered constructs

Fact versus artifact: Avoiding erroneous estimates of sulfated glycosaminoglycan content using the dimethylmethylene blue colorimetric assay for tissue-engineered constructs

Effects of cell type and configuration on anabolic and catabolic activity in 3D co‐culture of mesenchymal stem cells and nucleus pulposus cells

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